Reliable knowledge of manure nutrient content for intensive animal operations is imperative to development of effective comprehensive nutrient management plans, which will minimize nutrient runoff and pollution of adjacent waterways. The objectives of this study were to evaluate the spatial variation of manure dry-matter (DM), phosphorus (P), and nitrogen (N) content in commercial high-rise laying-hen houses, and to determine the sampling locations and number of samples that will lead to good assessment of nutrient content of manure in the houses. Two side-by-side manure samples were collected from 9 locations in each of 6 high-rise laying-hen houses (18 samples per house) and analyzed for DM, N, and P content. The 9 sample locations were distributed as one-fourth, half, and three-fourths of the building length, with 3 sample locations (every other manure row) per cross-section of the 5 manure rows. The average of DM, N, or P content from the 18 samples per house was used as the reference value for comparison of 8 sampling scenarios. Results showed that duplicate sampling at a location added little to the precision of the data. Manure samples collected crossways across the middle of the house or diagonally across the house in either direction yielded results most similar to the reference value for that house. Hence, when collecting manure samples for nutrient assessment in high-rise laying-hen houses, a single sample collected from every other manure pile across the middle of the building should be sufficient to obtain representative samples of the house and is recommended.